Mechanism of conjugation and recombination in bacteria. XX, Recombination DNA synthesis during mating in Escherichia coli K-12.

In crosses of dnaA recipients with HfrH at restrictive temperature a functional recombinant structure and viable recombinants were formed at a nearly normal rate, whereas recombination in crosses with HfrC was markedly inhibited. Recombinational DNA synthesis, as determined by 3H-thymine incorporation, was found in dnaA merozygotes from HfrH crosses. The recombinational synthesis was completed shortly after the end of mating. The replication of recombinant chromosomes in dnaA Lac+ recombinants started 120 minutes after interruption of mating, and at the restrictive temperature was sensitive to acridine orange, thus pointing to the presence of integrated F factor in the recombinant chromosome.     

Mechanism of conjugation and recombination in bacteria. XVII. Further evidence for single-stranded regions in recipient DNA during conjugation in Escherichia coli K-12.

The secondary structure of recipient DNA mated with Hfr strain was investigated by CsCl density gradient fractionation. After 45 min of HfrH64 X 3h-f-ab1157 mating one-fourth of the radioactive recipient DNA was recovered as a single-strand but only after shearing of cell lysates prior to centrifugation. This heavier than native DNA fraction of radioactive material (obtained after the first centrifugation) was degraded by single-strand specific nuclease S from Aspergillus oryzae. These findings thus confirm the authors' earlier results suggesting that in the course of mating are generated local single-stranded regions in recipient DNA.     

Scale of the genetic map and genetic control of recombination after conjugation in Escherichia coli K-12.

Summary There exist many regions on the genetic map of E. coli, remarkable for very high frequency of genetic exchanges between the donor and recipient chromosome after conjugation. We call these regions fre (frequent recombination exchange). Two of them were localized: frel near to the gene tsx and fre2 adjacent to metB. The conjugational transfer of fre is characterized by high negative interference in the corresponding region of the map.The effect called Fre is genetically determined. It is slightly present on the Rec BC pathway of recombination and becomes drastic on the Rec F pathway. The effect is sharpened by an increase of temperature till 43° C during and after conjugation. The effect is absolutely dependent on the genes recA and recF.It is assumed that region fre contains many hot spots of recombination, i.e. sites of initiation, where a recF-dependent endonuclease starts the process. The scale of the genetic map of E. coli K-12 in the areas not including the fre regions is about 24 min both on the Rec BC and the Rec F pathways. In the regions including fre, the saale drops to 5 min on the Rec BC pathway and to about 1 min on the Rec F pathway. These strong variations explain the discrepancies in the mapping distances found in different works.If a plasmid F' containing the fre region is transmitted during conjugation it becomes extremely unstable. A fragment of DNA containing the fre region is always lost from the plasmid. It leads to its shortening or sometimes to the killing of the cell. The Fre effect is seen also in P1 transduction. These facts pose many questions. Suggestive answers are discussed.     

Mechanism of conjugation and recombination in bacteria. XXI. Role of F factor genes in post-conjugational recombination in Escherichia coli K-12

Temperature sensitive dnaA recipient crossed at the restrictive temperature with HfrH, free from contaminating F+ cells, forms recombinants almost as proficiently as at the permissive temperature. The merozygotes are able to synthesize DNA at 42 degrees C, although the recipient and donor cells do not incorporate 3H-thymine. A substantial fraction of Lac+ recombinants, irrespective of the mating temperature, is temperature resistant (42 C-R); 15% from among those mated at 35 C and 30% from those mated at 42 C. The presence of dnaA mutation in these Lac+ 42 C-R recombinants was ascertained by co-transduction with ilv. Cell division at 42 C is inhibited in the Lac 42 C-R recombinants by acridine orange. The presence of F factor DNA in these recombinants was demonstrated directly by DNA: DNA hybridization. Suppression of dnaA mutation in Lac+ 42 degrees C-R recombinants and their sensitivity to acridine orange at 42 degrees C suggests that at least part of the F factor is integrated into the recombinant chromosome. A large fraction of the Lac+ 42 degrees C-R recombinants (up to 80%) is sensitive to male phage R17 and fertile. In crosses with HfrC there is a marked decrease of recombination frequency at 42 degrees C in the dnaA recipient. The fraction of Lac+ 42 degrees C-R recombinants is low (up to 10%) and the 42 degrees C-R recombinants are neither sensitive to male phage nor fertile. The results are discussed on the basis of the previously proposed model of post-conjugational recombination.     

Genetic control of recombination exchange frequency in Escherichia coli K-12.

The frequency of recombination exchanges per unit length of DNA (Freuld) can be estimated by measuring the scale of the genetic map that is the mean statistical distance between two neighboring crossovers. The scales appear to be equal for the alternative pathways of recombination, RecBCD (wild-type cells) or RecF (recBC- sbcB- sbcC- genotypes). The absolute value of the scale depends on specific experimental conditions. recR, recQ, ruv, recJ and recN genes of the RecF pathway of recombination (recBC- sbcBC- cell genotypes) do not appear to be silent in wild-type cells where the RecBCD pathway predominates. On the contrary, these genes are responsible for the Freuld. The list recF504::Kmr greater than recQ61::Tn3 greater than ruv-54 greater than recJ284::Tn10 shows decreasing efficiency in inhibiting recombination exchanges by these mutations. The recN264 mutation gives a small, but opposite effect of increasing the frequency of recombination exchanges. The effect of the recF and recQ mutations appears to be additive, but that is not the case in combinations of ruv-54 with recF504::Kmr or recQ61::Tn3.